We have previously shown that estrogen-stimulated transcriptional activity of human lactoferrin gene in endometrium carcinoma cells, RL95-2, is mediated through a functional imperfect estrogen response element (iERE) present in the 5' flanking region of the gene. Upstream from the iERE, a 36 bp DNA sequence (-418 to -378, FP1) is selectively protected by nuclear protein of the endometrium (RL95-2) and mammary gland (HBL100) cells from DNase I digestion. Three different nuclear proteins bind to the FP1 region and we have identified 2 of them, the COUP-TF and the hERR alpha1. From the electrophoresis mobility shift assay (EMSA), site-directed mutagenesis and DNA methylation interference analyses, we showed that binding of hERR alpha1 to an extended half site of the steroid receptor binding element in FP1 enhances ER-mediated transcriptional activity of the human lactoferrin promoter. Mutation at the Gs in this region abolishes hERR alpha1-DNA complex formation and reduces the estrogen responsiveness of the reporter plasmid containing the intact lactoferrin iERE. To examine the mechanism by which the hERR alpha1 and its DNA binding element enhance the estrogen response, we performed far-western analyses. The results showed that hERR1 interacted with estrogen receptor by direct protein-protein contact. These findings suggest that the hERR1 alpha1 modulates ER-mediated transcriptional activity by direct contact with the estrogen receptor. Recently, we cloned and characterized the hERR alpha gene. By Fluorescent In-Situ Hybridization method (FISH), the hERR alpha gene was localized to the centromere region of chromosome 11q12. Partial sequencing, restriction mapping and PCR analysis revealed that the hERR alpha gene consists of 7 exons and is over 20 kb in length. Al intron/exon junctions follow GT/AG rule. There are 2 well conserved intron/exon junctions (2 & 3) when compared with the human estrogen receptor. The highly conserved DNA binding domain of hERR alpha1 is present in exon 2 & 3. Primer extension and RNase protection analyses reveal multiple transcription initiation sites in different human cell lines. The sequence immediately 5' of the transcription start sites of hERR alpha1 message lacks a typical TATA box and CAAT box but is GC-rich and contains 10 putative Sp1 binding sites. The region that contains the multiple Sp1 binding sites showed high levels of promoter activity when transiently transfected into RL95-2 cells.